Received December 2, 2008; accepted January 12, 2009; available online January 15, 2009

Abstract: This study aims to improve the health of patients suffering from chronic fatigue syndrome (CFS) byinterventions based on the biochemistry of the illness, specifically the function of mitochondria in producing ATP(adenosine triphosphate), the energy currency for all body functions, and recycling ADP (adenosine diphosphate)to replenish the ATP supply as needed. Patients attending a private medical practice specializing in CFS werediagnosed using the Centers for Disease Control criteria. In consultation with each patient, an integer on the BellAbility Scale was assigned, and a blood sample was taken for the “ATP profile” test, designed for CFS and otherfatigue conditions. Each test produced 5 numerical factors which describe the availability of ATP in neutrophils,the fraction complexed with magnesium, the efficiency of oxidative phosphorylation, and the transfer efficienciesof ADP into the mitochondria and ATP into the cytosol where the energy is used. With the consent of each of 71patients and 53 normal, healthy controls the 5 factors have been collated and compared with the Bell AbilityScale. The individual numerical factors show that patients have different combinations of biochemical lesions.When the factors are combined, a remarkable correlation is observed between the degree of mitochondrialdysfunction and the severity of illness (P<0.001). Only 1 of the 71 patients overlaps the normal region. The “ATPprofile” test is a powerful diagnostic tool and can differentiate patients who have fatigue and other symptoms asa result of energy wastage by stress and psychological factors from those who have insufficient energy due tocellular respiration dysfunction. The individual factors indicate which remedial actions, in the form of dietarysupplements, drugs and detoxification, are most likely to be of benefit, and what further tests should be carriedout.

Introduction fatigue is considered to be a subjective

sensation characterized by lack of motivationChronic Fatigue Syndrome (CFS) is a and of alertness [1], even though the brain is amultisystem illness that robs its victims of their major consumer of resting cellular energy.health and their dignity. Two of the most Patients may demonstrate negative illnesscharacteristic and debilitating signs of CFS are beliefs that increase the severity of thevery poor stamina and delayed post-exertional symptoms [2, 3]. However, if the metabolism isfatigue. Sometimes the fatigue is mainly functioning properly, the fatigue and relatedmental, and sometimes mainly physical. symptoms must be due to energy beingFatigue is the same as lack of energy and wasted by the mental and physical processesenergy comes from the basic metabolic of stress, anxiety, tension and depression.process of the oxidation of food. Patients should be able to be helped, possibly cured by psychological intervention, e.g.A widely-held hypothesis (A) is that the cognitive behavioural therapy. In order tometabolism of people with CFS is normal, but explain the post-exertional malaise an ancillarythe fatigue and other symptoms are due to hypothesis (A') is needed, namelypsychological factors. It is acknowledged that deconditioning due to disuse of muscles.physical fatigue is lack of energy, but mental However, hypothesis A' is not supported by Chronic fatigue syndrome and mitochondrial dysfunction

experiment in many cases as we will see have also found severe deletions of genes inbelow. mitochondrial DNA (mtDNA), genes that are associated with bioenergy production [9, 10].An alternative hypothesis (B) is that there is a One consequence of mitochondrialmetabolic dysfunction with the result that not dysfunction is increased production of freeenough energy is being produced. The main radicals which cause oxidative damage. Suchsource of energy comes from the complete oxidative damage and increased activity ofoxidation of glucose to carbon dioxide and antioxidant enzymes has been detected inwater. The digestive system produces glucose, muscle specimens [11]. Some essentialglycerol and fatty acids, and amino acids. If compounds (carnitine and N-acylcarnitine)there is a problem with the digestive system, needed for some metabolic reactions ine.g. gut fermentation, hypochlorhydria or mitochondria have been measured in serumpancreatic insufficiency, energy production will and found to be decreased in patients withbe impaired and fatigue may result [4]. These CFS [12, 13]. Both studies found that theconditions can and should be tested for. carnitine levels correlated with functionalAllergies and thyroid malfunction can also capacity. Reduced oxidative metabolism [14-produce fatigue. 16] and higher concentrations of xenobiotics, lactate and pyruvate [17] have been reported.When the digestive system is functioning In one group of patients a decrease ofproperly glucose and lipids are fed into the intracellular pH after moderate exercise wasblood stream where, together with oxygen observed and a lower rate of ATP synthesisbound to hemoglobin in erythrocytes (red during recovery was measured [18]. Theseblood cells), they are transported to every cell findings suggest impaired recycling of ADP toin the body. In the cytosol of each cell glucose ATP in the mitochondria.is broken down in a series of chemicalreactions called glycolysis into two molecules However, there are also some similar studiesof pyruvate which enter the energy-producing that do not confirm mitochondrial dysfunction.organelles present in most cells of the body, This situation is likely due to the differentthe mitochondria. Some structural details and diagnostic criteria in use. For example, thethe number of mitochondria per cell are Oxford criteria [1], a definition proposed bydictated by the typical energy requirements; psychiatrists, require only fatigue; “othercardiac and skeletal muscle cells and liver and symptoms may be present” but are notbrain cells contain the highest numbers. The essential. The Centers for Disease Controlmitochondria generate energy by oxidative (CDC) criteria are more selective as theymetabolism in the form of ATP (adenosine require an additional four symptoms from a listtriphosphate) which when hydrolysed to the of eight [19]. In England in 2007 the Nationaldiphosphate, ADP, releases energy to produce Institute for Clinical Excellence (NICE)muscle contractions, nerve impulses and all introduced yet another set of criteria, fatiguethe energy-consuming processes including the plus one more symptom, for examplechemical energy needed to synthesise all of “persistent sore throat” [20]. At the other endthe complex molecules of the body [5, 6]. of the spectrum are criteria based on studiesThus, mitochondrial dysfunction will result in of patients with Myalgic Encephalomyelitisfatigue and can produce other symptoms of (ME) [21-23] which have culminated in theCFS. Canadian consensus criteria [24]; the Canadian criteria are unlikely to includeThe two hypotheses are not mutually patients satisfying only hypothesis A. Evenexclusive. Some patients may satisfy both. more confusingly, both the Canadian and theHowever there are constraints; the basal new NICE criteria use the term ME/CFSmetabolic rate (about 7000 kJ per day) must although their criteria are very different. At thebe maintained and the first law of present time the CDC criteria arethermodynamics must not be violated. internationally widely used as the criteria for research purposes despite their lack ofThere is considerable evidence that precision [25]. This situation may change inmitochondrial dysfunction is present in some the future because the Canadian criteria areCFS patients. Muscle biopsies studied by gaining wider acceptance and one charitableelectron microscopy have shown abnormal research funding agency (ME Research UK)mitochondrial degeneration [7-9]. Biopsies now requires both the CDC and Canadian

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criteria to be used in research projects that it (JMH), is designed specifically for CFS andfunds. We use the term CFS or CFS/ME for the other conditions where energy availability isCDC criteria and ME/CFS for the Canadian reduced. It was found early on that the “ATPcriteria. Our study is aimed to assess the role profile” was very useful in predicting the levelof mitochondrial dysfunction with the primary of disability and suggesting the most likelyaim of helping patients interventions which would benefit patients. Tests have now been carried out on a numberHypothesis B is attractive because of patients and also on normal, healthymitochondrial dysfunction in various organs subjects. When collated the test results showoffers possible explanations for many of the features that were completely unexpected.other symptoms of CFS and ME. There is Before we report here on the test proceduresmounting evidence that the symptoms are due and results we provide a brief summary of howto dysfunctions on the cellular level. mitochondria produce energy.Abnormalities have been seen in immune cells[26], and gene expression studies have Mitochondrial energy metabolismrevealed abnormalities in genes associatedwith immune cells, brain cells, skeletal muscle In each cell glucose is broken down tocells, the thyroid, and mitochondria [27, 28]. A pyruvate with the production of some ATP (2further genetic study identified seven clinical molecules net per molecule of glucose). Thephenotypes [29]. There seem to be three pyruvate and also fatty acids enter thedistinct clusters of clinical abnormalities that mitochondria of each cell, showndefine CFS [30]: (a) blood flow and vascular schematically in Figure 1, where twoabnormalities such as orthostatic intolerance coordinated metabolic processes take place:(vascular system), (b) widespread pain, and the tricarboxylic acid (TCA) cycle, also knownhigh sensitivities to foods, temperature, light, as the Krebs’ citric acid cycle, which producesnoise and odours (central nervous system some ATP, and the electron transport chainsensitization), and (c) fatigue, exhaustion and (ETC, also called the Respiratory Chainbrain fog (impaired energy production). because it uses most of the oxygen we breatheHypothesis B is that the lack of energy in the in) which regenerates ATP from ADP by thethird cluster originates in the mitochondria of process of oxidative phosphorylation (ox-phos).individual cells. But mitochondrial dysfunction Altogether some 30-odd molecules of ATP arecan also produce abnormalities (a) and (b) produced per molecule of glucose and thesebecause ATP produced in each cell by its constitute the main cellular energy packetsmitochondria is the major source of energy for used for all life processes. As well as food andall body functions. oxygen the metabolic pathways require all the nutrients involved in the production of theThese observations from biomedical research large number of enzymes which control theinto CFS are very encouraging, but how long do many biochemical reactions involved and allpatients have to wait before there is some real the cofactors needed to activate the enzymesprogress in ameliorating their symptoms? In a [31-33]. Most of the enzymes are coded byprivate medical practice which specializes in nuclear DNA (nDNA) in the cell’s nucleus and aCFS the primary goal is to make the patients few are coded by mtDNA. Some of thefeel and function better. Treatment is started enzymes rely on other organs. For example,by making use of the existing biomedical thyroid hormone is needed in the TCA cycle. Onknowledge to provide a basis of nutrition, the other hand hyperthyroidism can uncouplelifestyle management and pacing. Thyroid, the ox-phos process [34], so a thyroid problemadrenal and allergy problems are also can lead to fatigue and this can be tested for.addressed if they occur. Most patients improve The human body contains typically less thanwith these interventions. However, in many 100 g of ATP at any instant, but can consumecases the improvement is not as great as the up to 100 kg per day. Thus the recycling ox-patient and doctor would like. When one of us phos process is extremely important and it(SM) became aware of the commercial “ATP produces more than 90% of our cellularprofile” testing package it was thought that energy. The main features and processes arethis might be useful in predicting the level of illustrated in a simplified form in Figure 1disability and identifying any biochemical (further details can be found in all college-levellesions that were at fault. The “ATP profile” textbooks on biochemistry, e.g. [6], and intesting package, developed by one of us secondary school advanced-level biology

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Figure 1. Main stages and location of energy metabolism in a human cell (left), and simplified details of a mitochondrion showing the main metabolic cycles and the oxidative phosphorylation respiratory chain (right). The outer mitochondrial membrane is highly permeable whereas the inner membrane is permeable only to water and gases. Special carrier and Translocator proteins pass reactants through it. At the top are the proteins involved in the respiratory electron transfer chain (ETC) and in the transfer of ATP and ADP between the cytosol and mitochondrion. ADP and Pi are combined by ATP synthase to make ATP. The ADP/ATP Translocator opens OUT to transfer ADP into the matrix and opens IN to transfer ATP to the cytosol. Nicotinamide adenine dinucleotide plays a key role in its oxidised form NAD+ and its reduced form NADH + H+ in carrying and transferring protons (H+) and electrons (e-). Adapted from: [35] and [5].

textbooks, e.g. [35]). The ETC culminates with transfer outwards [37], and there is the the protein complex ATP synthase which is possibility that there may be other molecules effectively a reversible stepping motor in including environmental contaminants which which 3 ATP molecules are produced from can block transfers. ADP and inorganic phosphate (Pi) every revolution [36]. Because of evolutionary What happens if some part of these cellular history ATP is made inside the mitochondrial metabolic pathways goes wrong? If the inner membrane but used outside in the mitochondrial source of energy is cytosol where it releases energy by dysfunctional many disease symptoms may converting to ADP and Pi. The Pi as a appear [38] including the symptoms of CFS. negative ion is co-transported back inwards together with H+, while ADP3- is transported Suppose that the demand for ATP is higher inwards through the Translocator protein than the rate at which it can be recycled. This adenosine nucleotide translocase (TL or ANT) happens to athletes during the 100 meters in exchange for ATP4- moving out into the sprint. The muscle cells go into anaerobic cytosol. There are potential problems here metabolism where each glucose molecule is because it is known that some specific converted into 2 molecules of lactic acid. This molecules (e.g. atractyloside) block the process is very inefficient (5.2% energy transfer inwards and certain others can block production compared to the 100% of complete

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oxidation) and can last for only a few minutes. be performed. The laboratory carrying out theThe increased acidity leads to muscle pain. tests (Biolab Medical Unit, www.biolab.co.uk)Also, when the concentration of ADP in the was blinded to the Ability associated with anycytosol increases and the ADP cannot be blood sample. As tests were carried out onrecycled quickly enough to ATP, another more patients, it became clear that the “ATPchemical reaction takes place. This becomes profile” results were providing helpfulimportant if there is any mitochondrial information, and patients were asked to givedysfunction. Two molecules of ADP interact to written, informed permission for their test dataproduce one of ATP and one of AMP to be used anonymously. All patients have(adenosine monophosphate). The AMP cannot done this.be recycled [6] and thus half of the potentialATP is lost. This takes some days to replenish Blood samples from fifty-three normal, healthyand may account for the post-exertional volunteers were obtained by one of us (JMH)malaise symptom experienced by patients [39- as Laboratory Director of Biolab until43]. retirement from that position in 2007. Biolab obtained written permission with informedThus, mitochondrial dysfunction resulting in consent from each volunteer. The samplesimpaired ATP production and recycling is a from the patient group and the normalbiologically plausible hypothesis, and there is (control) group were processed in the sameconsiderable evidence that it is a contributory way. The control group consisted of 40factor in CFS, at least for a subset of patients. females of average age 36 (range 18 to 63)Our study may be considered to be a test of and 13 males of average age 35 (range 18 tothis hypothesis. 65).

Methods For both groups all procedures were consistent

with the Declaration of Helsinki (2000) of theParticipants World Medical Association (www.wma.net) and this report follows the guidelines of theSeventy-one patients, 54 female of average International Committee of Medical Journalage 47 (range 14 to 75) and 17 male of Editors (icmje.pdf available at www.icmje.org).average age 52 (range 20 to 86), wereselected from a total of 116 consecutive Procedurespatients attending a private medical clinicspecializing in CFS/ME. Patients were ATP is present in cells mainly as a complexexcluded only if they did not meet the CDC with magnesium and is hydrolysed to thediagnostic criteria for CFS [19] or if the “ATP diphosphate (ADP) as the major energy sourceprofile” test had been made before they had for muscle and other tissues. ADP conversionbeen seen clinically. Evaluations, tests and to ATP within mitochondria can be blocked orinterventions, where appropriate, were carried partially blocked by some environmentalout for diet and sleep problems, allergies, and contaminants. Specifically, the TL in thethyroid and adrenal problems. Advice on mitochondrial membrane that controls thepacing was also given. After this stage a transfer of ADP from the cytosol and ATP to themeeting was held with each patient at which cytosol may be chemically inhibited and itsan agreed numerical Ability was assigned and efficiency is also pH dependent. Changes inrecorded in the clinical notes. The integral CFS acid:base balance, magnesium status, and theAbility Scale [44] runs from 0 to 10 and is presence of abnormal metabolic products cangiven in Appendix A. It was proposed to those have similar effects to xenobiotic inhibition ofpatients who had not improved to an the TL.acceptable clinical level after theseinterventions that they have the “ATP profile” A number of methods have been developed fortest done. All the participating patients had assaying ATP. Methods such as magneticscores of 7 or less on the CFS Ability Scale. resonance spectroscopy (MRS) of 31P require the patient to be at a facility which is availableThe nature of the test was explained and each only at major hospitals or research institutes.patient agreed (and paid) for the “ATP profile” Biopsies of skeletal muscles can be taken, buttest (needing a 3-ml venous blood sample) to not of vital organs such as the heart, brain or

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liver. Methods using blood samples The mitochondria should then rapidly replete(specifically neutrophils) are relatively non- the ATP from ADP and restore the ATPinvasive and are amenable to routine testing. concentration. The overall result gives OxIn addition the blood stream reaches almost Phos, which is the ADP to ATP recyclingevery cell in the body and carries much efficiency that makes more energy available asinformation concerning what is going on. The needed.method of measuring ATP used in the “ATPprofile” dates from 1947 when McElroy (C). The TL switches a single binding sitepoured a solution of ATP onto ground-up firefly between two states. In the first state ADP istails and observed bright luminescence and recovered from the cytosol for re-conversion tofound that the amount of light produced was ATP, and in the second state ATP produced inproportional to the ATP concentration [45]. the mitochondria is passed into the cytosol toThus he showed that the energy contained in release its energy. Measurements are made byATP can produce light and this led the way to trapping the mitochondria on an affinitythe development of bioluminescent chromatography medium. First themeasurements which can be carried out mitochondrial ATP is measured. Next, an ADP-routinely and reproducibly with commercially containing buffer is added at a pH thatavailable biochemical assay kits and strongly biases the TL towards scavenging ADPbioluminescence equipment [46-49]. Light is for conversion to ATP. After 10 minutes theproduced when ATP reacts with D-luciferin and ATP in the mitochondria is measured. Thisoxygen in the presence of Mg2+ and the yields the number TL OUT. This is a measure ofenzyme luciferase. When ATP is the limiting the efficiency for transfer of ADP out of thereagent, the light emitted is proportional to the cytosol for reconversion to ATP in theATP present. mitochondria. In the next measurement a buffer is added at a pH that strongly biases theThe “ATP profile” test TL in the direction to return ATP to the cytosol. After 10 minutes the mitochondria are washedThe “ATP profile” test yields 5 independent free of the buffer and the ATP remaining in thenumerical factors from 3 series of mitochondria is measured and this gives themeasurements, (A), (B), and (C) on blood number TL IN. This is a measure of thesamples (neutrophils). Details of the efficiency for the transfer of ATP from themeasurements made and how the numerical mitochondria into the cytosol where it canfactors were calculated are given in Appendix release its energy as needed.B. The 3 series are: Results(A). ATP concentration in the neutrophils ismeasured in the presence of excess The individual numerical factorsmagnesium which is needed for ATP reactions.This gives the factor ATP in units of nmol per Figure 2 shows scatter plots (a point for eachmillion cells (or fmol/cell), the measure of how patient) of each of the 5 factors vs. CFS Ability.much ATP is present. Then a second As we will see later it is convenient to dividemeasurement is made with just endogenous the data from the 71 patients into 3magnesium present. The ratio of this to the categories, “very severe”, “severe”, andone with excess magnesium is the ATP Ratio. “moderate”, which have about the sameThis tells us what fraction of the ATP is number of entries (25, 21, and 25). To theavailable for energy supply. right of each scatter plot we show a stacked projection histogram for the 3 categories of(B). The efficiency of the oxidative Ability, and at the far right a histogram for thephosphorylation process is measured by first normal controls. Looking first at the ATPinhibiting the ADP to ATP conversion in the histogram for the normal controls we see alaboratory with sodium azide. This chemical well defined minimum value with a long tail upinhibits both the mitochondrial protein to a maximum value of 2.89 fmol/cell. Thecytochrome a3 (last step in the ETC) and ATP average value is 2.00 ± 0.05 (SEM (Standardsynthase [50]. ATP should then be rapidly used Error of the Mean), n=53) which can beup and have a low measured concentration. compared with the measurement, 1.9 ± 0.1Next, the inhibitor is removed by washing and (SEM, n=12), made some 25 years ago by there-suspending the cells in a buffer solution. same technique in a study of the energetics of

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Figure 2. Scatter plots of the 5 factors (A to E) measured in the “ATP profile” test vs. CFS Ability. In the middleare stacked projection histograms of the 3 categories of the patient group, and on the right projectionhistograms of the control group. The heavy horizontal dashed lines correspond to the minimum value of eachfactor measured for the control group.

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phagocytosis in neutrophils [51]. The stacked control group. Many patients, particularly thehistogram for the patients and the Ability plot “very severe”, are far below the normalclearly show that some patients are in the minimum.normal region and some are below and theysplit into two groups with very little overlap. The TL IN plot of Figure 2E also has a peak inRather than comparing the patients with the the normal region. However, some patientsaverage of the normal control group which is have very low values, including the patient withcustomary, we prefer to compare with the CFS Ability = 0. The product TL OUT × TL IN isminimum value of the control group which is only 0.012 for this patient who is very severelymore cleanly defined. Also, this method ill whereas this product is 0.17 at the normalpermits us to classify patients as being in the minima, a factor of 14 larger. If just ATP or Oxnormal region or being below the normal Phos had been measured the very severeregion. Clearly this can be changed easily and mitochondrial dysfunction of this patient wouldall the numbers are given in Figure 2. We not have been detected. Note the strongtherefore show as a heavy horizontal dashed positive correlation for TL IN.line the minimum value of each factormeasured for the controls. Most patients are below normal in more than one factor (average [range] is 3.7 [2 to 5] forFigure 2A, ATP vs. CFS Ability, shows that the “very severe”, 3.5 [2 to 5] for “severe” and 2.2majority of the “very severe” and “severe” [1 to 4] for “moderate”). Some of the featurespatients are below the normal minimum but are summarised numerically in Table 1.very few are below 75% of this minimum. Notethat 3 of the “very severe” patients are well For most of the factors the percentage ofinto the normal region; they have problems patients who are in the normal regionwith one or more of the 4 other factors. Just increases in going from “very severe” toover 50% of the “moderate” patients are in the “severe” and to “moderate”. The exception isnormal region. There is a small positive the ATP Ratio which gently decreases, butcorrelation which is indicated by the “trend” within the statistical errors is constant. Both TLcrosses. There is not a gentle increase in ATP IN and the product TL OUT × TL IN increase bywith Ability, but an increase in the fraction of large factors. For patients in the “moderate”patients above the normal minimum line. category the main influence on their illness appears to be the ATP Ratio.Figure 2B shows ATP Ratio vs. CFS Ability. Themajority of patients in all 3 categories are Table 1 also illustrates the importance ofbelow the normal minimum, and about 1/3 of measuring more than one factor. For example,“moderate” patients are below 75% of the if only ATP had been measured, 28% of all thenormal minimum. The correlation with Ability is patients would be classified as normal, and ifslightly negative. Values for the normal only Ox Phos had been measured, 32% of thecontrols are rather tightly grouped with a “very severe” patients would be classified asminimum of 0.65 and average of 0.69. normal.

The Ox Phos plot in Figure 2C shows a wide Correlations between numerical factorsrange of values and a strong positivecorrelation for this factor for the patient group. It is also helpful to look at correlationsThe stacked projection clearly shows that between pairs of numerical factors. The fivethere are two groups – above and below the most relevant examples are shown in Figure 3.normal minimum and the upper group spans asimilar range to the controls. Note the high In the scatter plots of Figure 3 the normalvalue for the sole patient with CFS Ability = 0. region is the rectangular region in the upperThis patient also has ATP = 1.26 and ATP Ratio right corner defined by the normal minima= 0.59 which are not very far below the normal dashed lines. In the ATP Ratio vs. ATP plotminima. (Figure 3A) most patients are fairly close to the normal region apart from the small cluster atThe TL OUT plot of Figure 2D also shows two ATP Ratio ~ 0.35.groups with a rather sharp peak in the stackedprojections just above the normal minimum In the Ox Phos vs. ATP plot (Figure 3B) thereand this closely matches the projection of the are only a few patients, all “moderate”, in the

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Table 1. Some features of the factors measured in the “ATP profile” tests

Percentage of patients in normal region

Category ATP ATP Ox Phos TL OUT TL IN TL OUT×TL IN Ratio

all (N=71) 28 ± 5 28 ± 5 38 ± 6 39 ± 6 52 ± 6 30 ± 5

very severe 12 ± 7 32 ± 10 32 ± 10 24 ± 9 24 ± 9 8±6

severe 19 ± 9 29 ± 10 19 ± 9 38 ± 11 48 ± 11 14 ± 8

moderate 52 ± 10 24 ± 9 60 ± 10 56 ± 10 84 ± 8 64 ± 10

The errors shown are ±1 SD (Standard Deviation), computed with the binomial distribution.

Figure 3. Scatter plots of correlations between pairs of factors measured in the “ATP profile”.

normal region for both factors. Some of the and vice versa. This is expected because the“very severe” and “severe” patients are in the ATP concentration is a major factor in thenormal region for Ox Phos and some are far control of the rate of the ox-phos process andbelow. Note the apparent negative correlation the energy supply is adjusted to meet thefor the normal controls. This shows that for energy demand. There is no obvious evidencenormal subjects there is a compensatory for this effect in the patient group.mechanism, i.e. if ATP is high Ox Phos is low

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In the Ox Phos vs. TL OUT plot (Figure 3C) only supply of ATP. Ox Phos is the efficiency of the6 (all “moderate”) patients are in the normal ETC which converts ADP into ATP. However, forregion of both factors. Note that as a function the recycling of ADP to make more energyof TL OUT there are 2 groups, a distinct narrow available the Translocator protein mustband to the right of the vertical dashed line efficiently have its binding site facing out toand a spread-out group to the left of this line. collect ADP (TL OUT) and alternately facing inLooking vertically, note that for the first group (TL IN) to efficiently transmit ATP from thethe Ability of patients (indicated by the 3 mitochondria into the cytosol where its energycategories) is correlated with their value of Ox can be used.Phos. There is a large spread in this factor andagain there appears to be two groups roughly We have found it useful to calculate thedivided by the horizontal normal minimum line. product of the five factors, the overallSome of the patients in all 3 categories are mitochondrial energy-producing relativeabove this line, but they have problems with efficiency, and call it the Mitochondrial Energyone or more of the other factors. Some of the Score. We just multiply the 5 factors together“very severe” patients have Ox Phos lower for each patient and each control. Thethan the normal minimum by an order-of- minimum value for the controls is 0.182magnitude. fmol/cell. We have chosen this as our normalisation point so we divide all the EnergyIn the Ox Phos vs. TL IN plot (Figure 3D) there Scores (for both patients and controls) by thisare many more patients in the normal region value. Thus all controls have Mitochondrialfor both factors, but also many with very low Energy Score ≥ 1.00.values of one or both factors. A scatter plot of the Energy Score for eachIn the TL IN vs. TL OUT plot (Figure 3E) there patient at each value of CFS Ability and eachare two clusters, one in the upper right corner control is shown in Figure 4A. The horizontalwhich is the normal region for both factors, dashed line indicates the minimum value forand another well below it at TL IN ~ 0.1. the normal controls and this is ourSeveral patients are far below the normal normalization value of 1.00. Only one of theminimum for both factors. 71 patients has an Energy Score > 1 (namely 1.25 for one of the patients with Ability = 7).In the biochemical methods used we might However this patient has 2 of the 5 factorsexpect some correlation between the TL below the normal minima.factors and Ox Phos because they are closelycoupled and interacting parts of the ADP to Note the high degree of correlation betweenATP reconversion cycle. However, the plots Energy Score and CFS Ability and this isindicate that the biochemical methods used independent of where the mean or minimumcan separate the Ox Phos and TL factors and of normal subjects is. It is natural to believemeasure them individually. that the CFS Ability of patients is more likely to depend upon mitochondrial dysfunction thanTo our knowledge this is the first time that vice versa, so we should really plot CFS Abilitysuch detailed effects have been observed. vs. Energy Score. However, the Ability was measured first, and Figure 4A showsThe Mitochondrial Energy Score convincingly that mitochondrial dysfunction is a major risk factor, and this has not beenThe biochemical measurements in the “ATP demonstrated before. Also shown in Figure 4Aprofile” separate the energy generation and is the best straight line fit to all 71 entries. Therecycling processes into 5 steps. As in any fit is good, but there is no reason that themultistep process, for example electrical relationship should be a straight line. Table 2power production or an assembly line, the gives the parameters of the fit. The Standardefficiency of the overall process is the product Error in the slope of the fitted straight line is soof the efficiencies of the individual steps. Any small that the probability P of the nullsuggestion of relative weighting is irrelevant; it hypothesis (i.e. that the slope is zero) isonly results in an overall normalization factor. extremely small, P <0.001, when computedThe product of ATP and ATP Ratio is the from the Student’s t-distribution [52]. Thecellular concentration of ATP complexed with 99.9% confidence interval is 0.092 < β <magnesium and this is the available energy 0.174 where β is the true slope and this lower

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Figure 4. The Mitochondrial Energy Score. A. The Energy Score plotted against CFS Ability with a point for each patient. A point for each control is plotted at CFS Ability = 10. The horizontal dashed line at Energy Score = 1.00 is our normalisation at the minimum Energy Score for controls. Also shown is the best straight line fit to the patient data. B. The Energy Score plotted vs. Age of patients and controls.

Straight line fit results

Slope 0.132± 0.012 Observed t-test 11.4

Intercept - 0.123± 0.042 Degrees of freedom 69†

R2 0.645* t-test probability, P < 0.001

*R2 (called the “coefficient of determination” or the “explained variation”) is

the square of the product moment correlation coefficient. † There are 71 data points and 2 parameters, slope and intercept.

limit is still several Standard Errors above zero. control group as compared to the patient group may influence our results. We haveIn Figure 4B the Energy Scores are plotted as a looked at the age dependence of all 5 factorsfunction of the age of each participant. It is and see no effect, and this is not surprising inbelieved that mitochondria play a major role in view of the wide spread in values of eachthe aging process [31, 33] so there is the factor. The Energy Score is a more reliablepossibility that the younger mean age of the measure of mitochondrial dysfunction. In

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Chronic fatigue syndrome and mitochondrial dysfunction

Figure 4B there is no evidence for age observed here.

dependence in the control group but themaximum age is only 65 years (points for three The ways that the individual factors in thecontrols have been omitted because their Mitochondrial Energy Score behave show thatEnergy Score is more than 2.00 with a not all patients are affected in the same way.maximum of 2.83). There are six patients of This may be due to the heterogeneous natureage ≥ 70, one “very severe” with Ability = 2, of the precipitating agents or to variations inone “severe” with Ability = 3, and four the way patients react to them. The results“moderate” who all have Ability = 4. These four indicate specific biochemical lesions and somepatients have Energy Scores which are below of these may be amenable to ameliorativethe average (0.42) for this Ability so there may intervention. Mitochondria need all of theirwell be a decrease with increasing age. On the essential vitamins, minerals, essential fattyother hand there is a 33-year old patient who acids and amino acids to function properlyalso has Ability = 4 and is well below the [31-33]. From the clinical point-of-view ofaverage. Excluding the six patients of age ≥ 70 helping patients this is very important; theslightly improves the straight line fit (R2 = typical time-consuming hit-or-miss protocol0.677) but has negligible effect on the other can be replaced by interventions based onparameters or our conclusions. biochemical information and understanding [53].Discussion An analysis of the outcomes of interventionsThe “ATP profile” results indicate being carried out (see www.drmyhill.co.uk)mitochondrial dysfunction of the neutrophils in will be the subject of a future publication.the patients in our cohort, and moreover thedegree of dysfunction is strongly correlated Conclusionswith the severity of their illness. Neutrophilsare the major effector cells of the immune We have demonstrated the power andsystem and the observed mitochondrial usefulness of the “ATP profile” test indysfunction is bound to have a deleterious confirming and pin-pointing biochemicaleffect on this system. We note that increased dysfunctions in people with CFS.apoptosis of neutrophils has been observedpreviously in people with CFS [26]. Our observations strongly implicateMitochondria are important functional parts of mitochondrial dysfunction as the immediatealmost all human cells but we cannot assert cause of CFS symptoms. However, we cannotfrom the present study that the mitochondria tell whether the damage to mitochondrialin other cells are dysfunctional to the same function is a primary effect, or a secondarydegree; human biology provides energy to vital effect to one or more of a number of primaryorgans at the expense of less important parts. conditions, for example cellular hypoxia [30],However, dysfunction in heart muscle cells or oxidative stress including excessiveand in central nervous system cells could peroxynitrite [54-58]. Mitochondrialexplain respectively the vascular and central dysfunction is also associated with severalsensitization clusters of clinical abnormalities other diseases and this is not surprising inmentioned in the Introduction. Thus, our view of the important role of mitochondria inresults strongly suggest that the immediate almost every cell of the body, but this factcause of the symptoms of CFS/ME is appears to have been recognised only inmitochondrial dysfunction. recent years [34, 38, 59, 60].

We cannot overemphasize the importance of a The observations presented here should be

careful diagnosis using the CDC criteria [19], confirmed in a properly planned and fundedor even better the Canadian criteria which study. The biochemical tests should be donemore precisely describe the symptoms [24]. on CFS patients after, as well as before,(An abridged version designed for health appropriate interventions and possibly onprofessionals, patients and carers is available patients with other disabling fatigueat www.mefmaction.net/Patients/Overviews/ conditions. It would also be good to confirmtabid/122/Default.aspx). It is doubtful that the biochemical test results in a secondpatient selection with less selective criteria (perhaps government-supported) laboratory.would yield the high degree of correlation

12 Int J Clin Exp Med (2009) 2, 1-16

Chronic fatigue syndrome and mitochondrial dysfunction

Acknowledgements exertion. Able to work full-time with difficulty.

We acknowledge helpful comments from Dr. 8. Mild symptoms at rest. Symptoms worsened byDerek Pheby and Dr. Neil Abbot. exertion. Minimal activity restriction noted for activities requiring exertion only. Able to work full time with difficulty in jobs requiring exertion.Address correspondence to: Norman E. Booth, PhD,‘Applegate’, Orchard Lane, East Hendred, Wantage 9. No symptoms at rest; mild symptoms withOX12 8JW, UK, Telephone: +44 (0)1235 833486 activity; normal overall activity level; able to workE-mail: n.booth1@physics.ox.ac.uk full-time without difficulty.Appendix A - The Bell CFS Ability scale 10. No symptoms at rest or with exercise; normal overall activity level; able to work or do house/homeThis scale is a useful and sensitive measure of the work full time without difficulty.level of activity and ability to function of patientswith CFS/ME [44]. It is similar to the Energy Index Appendix B - The “ATP profile” testsPoint Score (EIPS™, www.cfsviraltreatment.com)[61]. It runs from 0 to 10 with: The “ATP profile” tests were developed and carried out at the Biolab Medical Unit, London, UK0. Severe symptoms on a continuous basis; (www.biolab.co.uk), where one of us (JMH) wasbedridden constantly; unable to care for self. Laboratory Director until retirement in 2007. Blood samples in 3-ml heparin tubes were normally1. Severe symptoms at rest; bedridden the majority received, tested and processed within 72 hours ofof the time. No travel outside of the house. Marked venepuncture. We briefly describe here the 3 seriescognitive symptoms preventing concentration. of measurements, (A), (B) and (C) and how the 5 numerical factors are calculated. (Step-by-step2. Moderate to severe symptoms at rest. Unable to details can be obtained by contacting JMH atperform strenuous activity. Overall activity 30-50% acumenlab@hotmail.co.uk).of expected. Unable to leave house except rarely.Confined to bed most of day. Unable to concentrate Neutrophil cells are separated by HistopaqueTMfor more than 1 hour per day. density gradient centrifugation according to Sigma® Procedure No. 1119 (1119.pdf available at3. Moderate to severe symptoms at rest. Severe www.sigmaaldrich.com). Cell purity is checked usingsymptoms with any exercise; overall activity level optical microscopy and cell concentration isreduced to 50% of expected. Usually confined to assessed using an automated cell counter.house. Unable to perform any strenuous tasks. Able Quantitative bioluminescent measurement of ATP isto perform desk work 2-3 hours per day, but made using the Sigma® Adenosine 5’-triphosphaterequires rest periods. (ATP) Bioluminescent Somatic Cell Assay Kit (FLASC) according to the Sigma® Technical Bulletin4. Moderate symptoms at rest. Moderate to severe No. BSCA-1 (FLASCBUL.pdf). In this method ATP issymptoms with exercise or activity; overall activity consumed and light is emitted when fireflylevel reduced to 50-70% of expected. Able to go out luciferase catalyses the oxidation of D-luciferin. Theonce or twice per week. Unable to perform light emitted is proportional to the ATP present, andstrenuous duties. Able to work sitting down at home is measured with a Perkin-Elmer LS 5B3-4 hours per day, but requires rest periods. Fluorescence Spectrometer equipped with a flow- through micro cell. Sigma® ATP Standard (FLAA.pdf)5. Moderate symptoms at rest. Moderate to severe is used as a control and as an addition-standard forsymptoms with exercise or activity; overall activity checking recovery. Similar kits are available fromlevel reduced to 70% of expected. Unable to other providers, e.g. the ENLITENTM ATP Assayperform strenuous duties, but able to perform light System (Technical Bulletin at www.promega.com),duty or desk work 4-5 hours per day, but requires and dedicated instruments are now available, e.g.rest periods. Modulus Luminescence Modules (see Application Note www.turnerbiosystems.com/doc/appnotes6. Mild to moderate symptoms at rest. Daily activity /PDF/997_9304.pdf).limitation clearly noted. Overall functioning 70% to90%. Unable to work full time in jobs requiring (A). ATP is first measured with excess magnesiumphysical labour (including just standing), but able to added via Sigma® ATP Assay Mix giving result a.work full time in light activity (sitting) if hours This is the first factor, the concentration of ATP inflexible. whole cells, ATP = a in units of nmol/106 cells (or fmol/cell).7. Mild symptoms at rest; some daily activitylimitation clearly noted. Overall functioning close to The measurement is repeated with just the90% of expected except for activities requiring endogenous magnesium present by using